The cannabinoid receptor one (CB1) is a G protein-coupled receptor (GPCR) that binds the main psychoactive component of marijuana, A9-tetrahydrocannabinol. The CB1 receptor exerts its effects by binding to Gai/o G-proteins, resulting in the activation of effector pathways. Desensitization and internalization of the receptor govern the rate and magnitude of CB1 receptor activation. Both processes, for most non-visual GPCRs, are initiated following (3-arrestin binding, yet the mechanisms of a (3-arrestin-CB1 interaction, as well as the consequences on receptor trafficking, are poorly understood. The overall objectives of this project is to determine the p-arrestin subtype(s) that bind to the CB1 receptor, identify critical residues and structural features of the CB1 receptor that are involved in p-arrestin binding, and examine the cellular consequences of such an interaction. In Aim 1 of this proposal, the structural requirements of the CB1 receptor that are critical for binding purified P-arrestin(s) and the optimal binding environment will be delineated, using a combination of biochemical and NMR studies. Affinities of both p- arrestin subtypes for the CB1 receptor will be measured and will elucidate the preferred binding partner. To examine the consequences of CB1-p-arrestin interactions, in Aim 2 the subcellular distribution of p-arrestin and the full-length CB1 receptor will be examined individually and when co-expressed, in HEK293 and primary hippocampal neurons using confocal microscopy. In Aim 3, the role of structural motifs of the CB1 receptor critical for P-arrestin binding (identified in Aim 1) will be assessed in the full-length receptor using targeted mutatgenesis and the consequences of these mutations on receptor trafficking examined. These studies will improve our understandings of the mechanisms involved in desensitization and internalization of cannabinoid receptors, and by elucidating the exact nature of the CB1-P-arrestin complex, may elucidate novel drug targets. Relevance: The cannabinoid receptor one (CB1) is primarily distributed in the central nervous system. Agonist activation of the CB1 receptor has been shown to cause an increase in appetite, produce anxiety, sedation, and hypothermia, as well as be detrimental to learning and memory, whereas inverse agonist blockade results in appetite suppression and a decreased craving for drugs. The goal of this project is to elucidate CB1- beta arrestin interactions that lead to a functional decrease in CB1 and consequently impact the availability of receptors for therapeutic strategies.